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An immunological assay detects and quantifies acetylamantadine in the
urine or other bodily fluids of a person who has taken a small dose of
amantadine. The quantification of acetylamantadine can be used to
quantify SSAT activity and elevated SSAT activity is an indication of
diseases including, but not limited, to inflammations and cancers.

Inventors:

Cheng; Brian; (Chesterfield, MO)

Applicant:

Name

City

State

Country

Type

BIOMARK TECHNOLOGIES INC

Richmond

CA

Assignee:

BioMark Technologies Inc.RichmondBC

Family ID:

1000001872931

Appl. No.:

14/915616

Filed:

August 29, 2014

PCT Filed:

August 29, 2014

PCT NO:

PCT/CA2014/050833

371 Date:

February 29, 2016

Related U.S. Patent Documents

Application Number

Filing Date

Patent Number

61871642

Aug 29, 2013

Current U.S. Class:

435/7.93

Current CPC Class:

G01N 33/5308 20130101

International Class:

G01N 33/53 20060101 G01N033/53

Claims

1. A method of detecting acetylamantadine in bodily fluids of a mammal,
the method comprising: dosing the mammal with amantadine; taking a bodily
fluid sample from the mammal; incubating the bodily fluid sample with a
known concentration of antibodies specific for acetylamantadine; and
detecting acetylamantadine in the bodily fluid sample based on the
inhibition of the binding of anti-acetylamantadine antibodies to
acetylamantadine bound on a substrate.

2. A method of quantifying acetylamantadine in bodily fluids of a mammal,
the method comprising: dosing the mammal with amantadine; taking a bodily
fluid sample from the mammal; incubating the bodily fluid sample with a
known concentration of antibodies specific for acetylamantadine; and
quantifying acetylamantadine in the bodily fluid sample based on the
inhibition of the binding of anti-acetylamantadine antibodies to
acetylamantadine bound on a substrate.

Description

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to methods and compositions for the
quantification of spermine/spermidine N.sup.1-acetyltransferase (SSAT)
enzymatic activity.

[0003] 2. Description of the Related Art

[0004] Spermidine/spermine N.sup.1-acetyltransferase (SSAT) is
ubiquitously distributed in mammalian tissues and plays a role in
catabolism and elimination of polyamines from cells. SSAT is an inducible
enzyme that catalyzes the transfer of an acetyl group from an
acetyl-coenzyme A to the aminopropyl moiety of the polyamines. This
action by SSAT facilitates polyamine degradation, excretion, and cycling
and/or intracellular cycling. In this manner SSAT participates in the
maintenance of polyamine homeostasis in mammalian cells. However, in
normal or uninduced mammalian tissues SSAT is present at very low levels.

[0005] Induction of SSAT expression can be caused by different drugs,
growth factors, polyamines, polyamine analogues, toxic substances,
hormones and physiological stimuli. Although all of the aforementioned
compounds could cause induction of SSAT expression, induction occurs at
different times for each individual compound. The regulation of SSAT
expression occurs at the levels of transcription, mRNA stability, mRNA
translation and protein stability. Induction or over-expression of SSAT
is usually required for there to be sufficient SSAT enzyme present in
cells or 100,000.times.g supernatant before in-vitro experiments can be
successfully undertaken.

[0006] While current literature teaches that SSAT is an acetylating enzyme
specifically for substrates including spermine and spermidine or its
analogues, SSAT activity, SSAT enzyme kinetics and assay methodology for
non-spermine/spermidine substrates of SSAT have not been understood.
Current methods exist to quantify SSAT activity. However, these
techniques are dependent on highly skilled personnel and complicated
experimental methods. More specifically, there has been a need for assay
methodology which quantifies the activity of SSAT through detection of
acetylated forms of non-spermine. Spermidine substrates of SSAT,
including amantadine, may be used to detect various pathological
conditions.

[0007] Traditional methods such as Gas Liquid Chromatography (GLC), High
Pressure Liquid Chromatography (HPLC) alone or coupled with mass
spectroscopy are being used for assaying acetylated metabolite such as
acetylamantadine. The detection sensitivity requires parts per billion of
the target analyte which becomes a challenge. GLC and HPLC have been
shown to be effective for in-vitro assays but may not be practical for
in-vivo assays. Employment of deuterated analyte as an internal standard
for HPLC-MS-MS method to assay acetylamantadine was successful.

[0008] These traditional methods require labor intensive analytical
service resulting in high operating costs and capital costs. All of these
add to the inefficiencies of the healthcare economy. In addition,
biological samples must be logistically handled and shipped to a
centralized laboratory for assay. These operations may result in sample
quality changes and could result in false interpretation of the result.

SUMMARY OF THE INVENTION

[0009] It is an object of the present invention to provide an
immunological assay that detects, and preferably quantifies,
acetylamantadine in urine or other bodily fluids of a mammal or patient
given a small dose of amantadine.

[0010] It is disclosed that it is possible to inhibit, with a sample of
urine containing acetylamantadine, the binding of anti-acetylamantadine
polyclonal antibodies to acetylamantadine bound to a substrate.
Polyclonal antibodies that specifically recognize acetylamantadine are
generated by immunizing warm-blooded animals with an immunogen
acetylamantadine cross-linked to a carrier protein or a universal T cell
epitope preferably with an adjuvant. The methods of cross-linking
proteins, universal T cell epitopes, and adjuvants are conventional. The
polyclonal antibodies that specifically recognize acetylamantadine are
absorbed with amantadine because there are high concentrations of
amantadine in the urine of a mammal or patient who has taken a small dose
of amantadine. This can be done by conventional methods, such as putting
the antibodies on a column of agarose beads covalently coupled with
amantadine and taking the flow-through from the column. Also provided is
a quantitative assay with a standard curve with measured amounts of
acetylamantadine.

[0011] The form of in-vitro testing diagnostic (IVD) based on this
disclosure is an immunological assay that detects, and may quantify,
acetylamantadine at the clinical office. This may allow for quick medical
decisions and may indirectly measure SSAT activity in the body. A test
methodology at the point of care is therefore desirable to minimize
sample instability and reduce healthcare costs. The form of IVD at the
clinical office may allow quick medical decisions.

[0012] There may be designs of IVD based on this disclosure of an
immunological assay that measures acetyl amantadine in the urine of a
mammal who has taken a small dose of amantadine.

[0013] The polyclonal antibodies specific for acetylamantadine which are
absorbed by amantadine may be used to detect or quantify acetylamantadine
in the presence of an excess of amantadine.

[0014] The polyclonal antibodies specific for acetylamantadine which are
absorbed by amantadine may be used to detect or quantify
spermine/spermidine N.sup.1-acetyltransferase (SSAT) activity in the
body.

BRIEF DESCRIPTIONS OF DRAWINGS

[0015] The invention will be more readily understood from the following
description of the embodiments thereof given, by way of example only,
with reference to the accompanying drawings, in which:

[0016] FIG. 1 is a graph illustrating polyclonal affinity-purified
antibodies against acetylamantadine absorbed with amantadine inhibited by
small concentrations of acetylamantadine in a standard curve;

[0017] FIG. 2 is a graph illustrating polyclonal affinity-purified
antibodies against acetylamantadine absorbed with amantadine inhibited by
small concentrations of acetylamantadine and large concentrations of
amantadine;

[0018] FIG. 3 is a graph illustrating polyclonal affinity-purified
polyclonal antibodies against acetylamantadine inhibited by small
concentrations of acetylamantadine and large concentrations of
amantadine;

[0019] FIG. 4 is a graph illustrating serum from a rabbit inhibited by
small concentrations of acetylamantadine and large concentrations of
amantadine; and

[0020] FIG. 5 is a graph illustrating the results of an acetylamandine
competitive ELISA with signal reading against acetylamantadine
competitive concentration.

BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Disclosed herein is an immunological assay that may be used to
detect or quantify acetylamantadine in a mammal who has taken a small
dose of amantadine.

[0022] A bodily fluid sample is taken from the mammal and incubated with a
known concentration of polyclonal antibodies specific for
acetylamantadine. The acetylamantadine in the sample will inhibit the
polyclonal antibodies specific for acetylamantadine from binding to
acetylamantadine attached to a substrate. There are many ways to attach
acetylamantadine to the substrate where the antibody signal is detected.
Biotinylated acetylamantadine was attached to streptavidin attached to
the substrate in the example disclosed herein. The substrate is where the
signal is detected when anti-acetylamantadine antibodies bind to
acetylamantadine if the anti-acetylamantadine antibodies are not
inhibited by acetylamantadine in the sample. If the concentration of
acetylamantadine in the sample is exceeding the threshold of abnormal
concentrations, the signal on the substrate may be completely inhibited.
It would be understood by a person skilled in the art that many designs
of in-vitro testing diagnostic (IVD) that detects acetylamantadine in a
sample may be used. For example, coloured particles, like gold particles,
are bound to acetylamantadine and abnormal concentrations of
acetylamantadine in the sample inhibit the binding of coloured particles
to a test line bound with anti-acetylamantadine antibodies.

Preparation of Acetylamantadine Protein Conjugates

[0023] A conjugate of a carrier protein ovalbumin is linked by
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to an
amine-derivative of acetylamantadine using the manufacturer's
instructions. In this example, the manufacturer was Thermo Fisher
Scientific Inc. of 3747 N Meridian Rd, Rockford, Ill., United States of
America, 61101. Another immunogen that rabbits were immunized with was a
conjugate of keyhole-limpet hemocyanin linked by formaldhyde to an
amine-derivative of acetylamantadine. Another immunogen that rabbits were
immunized with was a conjugate of the biotin-binding protein avidin
coupled to an excess of biotinylated 4-amino-1-N-acetylamantadine. The
biotinylation was carried out using standard methods and instructions
from the manufacturer which, in this example, was Thermo Fisher
Scientific Inc. of 3747 N Meridian Rd, Rockford, Ill., United States of
America, 61101.

[0024] The amine-derivative of acetylamantadine was synthesized as
described in the following synthetic scheme.

Synthesis of Adamantylamine Derivatives

##STR00001##

[0025] Immunization of rabbit:

[0026] A New Zealand white rabbit was immunized intramuscularly with 250
.mu.g of the acetylamantadine-ovalbumin conjugate emulsified in complete
Freund's adjuvant. The rabbit was boosted multiple times with
acetylamantadine-ovalbumin conjugate, twice with 100 .mu.g of
acetylamantadine-conjugate keyhole-limpet hemocyanin and once with 4 pg
of avidin coupled with an excess of biotinylated acetylamantadine with
alum as an adjuvant. The rabbit was boosted with 100 .mu.g of the
acetylamantadine-ovalbumin conjugate 14 days before the rabbit was
euthanized and bled out.

Preparation of Immunoglobin G (IgG):

[0027] The rabbit blood was allowed to clot and the sera were collected.
The IgG was collected using beads coated with Protein A and the IgG was
eluted from the column by pH 2 glycine buffer and neutralized to pH 7.2.

Preparation of columns:

[0028] Cyanogen-bromide coupled agarose beads were conjugated with either
an amine derivative of acetylamantadine or an amine derivative of
amantadine.

Affinity-purified antibodies against acetylamantadine:

[0029] The IgG was flowed over a column of cyanogen-bromide coupled
agarose beads conjugated with acetylamantadine and the antibodies against
acetylamantadine bound to the column. The column was washed extensively
with phosphate buffered saline. The affinity-purified antibodies against
acetylamantadine were eluted from the column by pH 2 glycine buffer and
neutralized to pH 7.2.

[0030] The affinity-purified antibodies against acetylamantadine were
flowed over a column of cyanogen-bromide coupled agarose beads conjugated
with amantadine and the peak of IgG was taken.

Quantifying acetylamantadine with affinity-purified antibodies against
acetylamantadine absorbed with amantadine:

[0031] Enzyme-linked immunosorbent assay (ELISA) plates were coated with
the biotin-binding protein streptavidin at 2 .mu.g/mL and blocked with
skim milk. The ELISA plates were then washed with phosphate-buffered
saline with an automated washer. Biotinylated acetylamantadine at 2 ng/mL
was added to the streptavidin-coated ELISA plates so that
acetylamantadine was bound to the streptavidin. The ELISA plates were
then washed to remove the free biotinylated acetylamantadine. The soluble
acetylamantadine or the amatidine was titrated in a separate 96-well
plate and incubated for an hour with a small, previously determined
concentration of anti-acetylamantadine affinity-purified antibodies
absorbed with amantadine that gives a clear signal in the ELISA plates
coated with acetylamantadine. The smallest amount of affinity-purified
antibodies against acetylamantadine absorbed with amantadine gives the
greatest sensitivity for acetylamantadine. Then the titrations of
acetylamantadine and amantadine incubated for one hour with a constant
amount of anti-acetylamantadine affinity-purified antibodies absorbed
with amantadine were added to the ELISA plates coated with
acetylamantadine.

[0032] The ELISA was incubated for two hours and then the ELISA was
developed by adding goat antibodies against rabbit IgG conjugated with
alakine phosphatase. The substrate was added and the ELISA developed by
standard methods and the optical densities were assayed by an ELISA
reader and are shown in FIG. 1. In particular, FIG. 1 shows a standard
curve showing inhibition from 100 pg/mL from 10 ng/mL. If one makes
quadruplicate assays of samples of urine, one can accurately quantify
acetylamantadine in the assay. If the urine sample inhibits 100%, one can
make a dilution of the sample and accurately quantify acetylamantadine in
the assay. There was significant inhibition (50%) of the binding in the
ELISA with .about.400 pg/mL of soluble acetylamantadine added to the
anti-acetylamantadine affinity-purified antibodies absorbed with
amantadine as shown in FIG. 2. In contrast, there was significant
inhibition (50%) with only 40 .mu.g/mL of amantadine as shown in FIG. 2.

[0033] The affinity-purified antibodies from this individual rabbit that
bound to acetylamantadine, and not absorbed with amantadine, also worked
in this immunological assay as shown in FIG. 3. There was significant
inhibition (50%) of the binding in the ELISA with .about.1.3 ng/ mL of
soluble acetylamantadine added to the anti-acetylamantadine
affinity-purified antibodies absorbed with amantadine as shown in FIG. 2.
In contrast there was significant inhibition (50%) with only 100 .mu.g/mL
of amantadine as shown in FIG. 2.

Quantifying acetylamantadine with serum of a rabbit immunized repeatedly
against acetylamantadine:

[0034] The sera from this individual rabbit also worked in this
immunological assay that quantified acetyl amantadine as shown in FIG. 4.
There was significant inhibition (50%) of the binding in the ELISA with
.about.300pg/ mL of soluble acetylamantadine added to the dilution of
serum as shown in FIG. 2. In contrast, there was significant inhibition
(50%) with 100 .mu.g/mL of amantadine as shown in FIG. 2.

[0035] It will accordingly be understood by a person skilled in the art
that the immunological assay disclosed herein may be used to detect or
quantify acetylamantadine in a patient who has taken a small dose of
amantadine. This is shown in FIG. 5 which illustrates the results of an
acetylamandine competitive ELISA with signal reading against
acetylamantadine competitive concentration. The ELISA plate coating was 2
ug/ml streptavidin and 6 ng/ml biotinylated acetylamantadine. The
competition ELISA using serum at 1:200 dilution against acetylamantadine
ranging from 0.025 ng/ml to 250 ng/ml. Complete inhibition inhibition
occurs near 100 ng/ml of soluble acetylamantadine. The detection limit
(sensitivity) is near 0.5 ng/ml to 1.0 ng/ml The quantification of
acetylamantadine can be used to quantify SSAT activity and elevated SSAT
activity is an indication of diseases including, but not limited, to
inflammations and cancers.

[0036] It will still further be understood by a person skilled in the art
that many of the details provided above are by way of example only, and
are not intended to limit the scope of the invention which is to be
determined with reference to the following claims.